Quinoa willd agrobacterium rhizogenes transformation method and application

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a chenopodium quinoa willd agrobacterium rhizogenes transformation method and application, the method comprises the following steps: sterilizing chenopodium quinoa willd seeds, and culturing and germinating on a culture medium to obtain chenopodium quinoa willd seedlings; the method comprises the following steps: cutting part of quinoa seedlings, culturing on a culture medium, immersing into a culture medium with agrobacterium rhizogenes, culturing, transferring to sterilized filter paper, absorbing bacterial liquid on the surface, transferring to a culture medium on which a layer of sterile filter paper is paved, culturing, and screening the cultured quinoa plants. The method is simpler to operate and lower in technical threshold and cost, the T-DNA fragment of the Ti plasmid of the agrobacterium rhizogenes can be efficiently integrated into the chenopodium quinoa willd genome, the integration efficiency is higher than that of some chemical or physical transformation methods, stable inheritance and expression can be achieved, and the genetic transformation efficiency can reach 30%-50%.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a method and application of quinoa transformation using Agrobacterium rhizogenes. Background Art

[0002] Quinoa (Chenopodium quinoa Willd.), a highly nutritious and adaptable pseudocereal crop, has garnered widespread attention worldwide in recent years. However, quinoa breeding and gene function research face numerous challenges. First, quinoa's flowers are small and complex, making traditional hybridization methods difficult, time-consuming, and inefficient. Second, research on quinoa's gene function is relatively underdeveloped, partly due to the lack of efficient gene transformation systems, making in-depth analysis of quinoa's gene functions challenging.

[0003] Currently, the quinoa transformation technologies available on the market are mainly based on Agrobacterium-mediated genetic transformation methods. These methods all target the stem, root, and other parts of the plant, and are performed during the quinoa seedling stage. Although these methods have achieved genetic transformation of quinoa to a certain extent, they still have significant shortcomings. Specifically, the existing technologies have the following drawbacks: Low transformation efficiency: Many similar products have low transformation efficiency on quinoa, and some even fail to transform, which seriously limits the in-depth and extensive application of quinoa transgenic research.

[0004] Complicated operation steps: The operation steps of existing technologies are relatively complicated, involving multiple complex culture and processing processes, which increases the difficulty and cost of the experiment and reduces the efficiency of research.

[0005] Limited applicable varieties: Currently, most quinoa transformation technologies are only applicable to a few quinoa varieties, which greatly limits the promotion and application scope of the technology.

[0006] It's worth noting that while the CRISPR / Cas9 gene editing system has demonstrated tremendous potential in other crops, revolutionizing gene function research and crop breeding, its application in quinoa has yet to be reported. The lack of this advanced gene editing tool complicates gene function research and precision breeding in quinoa.

[0007] Quinoa's regeneration system is one of the keys to successful genetic transformation. However, there are currently few reports on successful quinoa regeneration, and none of these reports involve the genetic transformation process. This lack of a regeneration system makes stable genetic transformation in quinoa particularly difficult, limiting the development of molecular breeding for quinoa.

[0008] To address these challenges and shortcomings, the present invention aims to develop an efficient, simple, and adaptable Agrobacterium rhizogenes transformation method for a wide range of quinoa varieties. By optimizing quinoa callus induction conditions and establishing a regeneration system, the present technology is expected to address the challenges of existing quinoa transformation technologies, such as low transformation efficiency, cumbersome procedures, and limited applicability. Furthermore, the successful application of the present technology will provide strong support for gene function research and molecular breeding in quinoa, promoting the sustainable development of the quinoa industry. Summary of the Invention

[0009] The purpose of the present invention is to provide a quinoa rhizogenes Agrobacterium transformation method and application, which optimizes the quinoa callus induction conditions and establishes a genetic regeneration system, providing strong support for quinoa gene function research and molecular breeding, and effectively promoting the sustainable development of the quinoa industry.

[0010] The object of the present invention is achieved through the following technical solutions: The present invention provides a method for transforming quinoa with Agrobacterium rhizogenes, comprising the following steps: (1) sterilizing quinoa seeds and then culturing and germinating them on a culture medium to obtain quinoa seedlings; (2) Cutting parts of quinoa seedlings and culturing them on a culture medium; (3) After culturing in step (2), immersing the culture medium containing Agrobacterium rhizogenes for culturing; (4) After culturing in step (3), the cells are transferred to sterilized filter paper to absorb the bacterial liquid on the surface, and then transferred to a culture medium covered with a layer of sterile filter paper for culturing; (5) The quinoa plants cultured in step (4) are screened to obtain the obtained product.

[0011] Furthermore, in step (1), the disinfection treatment is performed using sodium hypochlorite, the culture medium is 1 / 2MS culture medium containing 3% sucrose, and the culture time is 3-4 days.

[0012] Furthermore, in step (2), the cutting is performed by cutting the hypocotyl of the quinoa seedling with a sterilized blade, and the culturing is performed by culturing the cut shoot part on a 1 / 2 MS medium containing 3% sucrose for 4-5 days.

[0013] Furthermore, in step (3), the culture medium containing Agrobacterium rhizogenes is LB liquid culture medium, and the culture is carried out at 28° C. and 200 rpm for 20 minutes.

[0014] Furthermore, in step (4), the culture medium is 1 / 2MS culture medium containing 3% sucrose.

[0015] Furthermore, in step (5), the screening is to detect whether the transformed specific gene is expressed.

[0016] The present invention also provides a quinoa plant prepared by the quinoa Agrobacterium rhizogenes transformation method.

[0017] The present invention also provides an application of the quinoa plant in the gene function research and molecular breeding of quinoa.

[0018] The beneficial effects of the present invention are: Compared with some complicated plant transformation methods, such as gene gun transformation, which requires expensive equipment and complex operations, the Agrobacterium transformation only requires co-cultivation of the explant with Agrobacterium rhizogenes containing the target gene, which is simpler to operate, and has lower technical barriers and costs. And Agrobacterium rhizogenes can efficiently integrate the T-DNA fragment of its own Ti plasmid into the quinoa genome, and the integration efficiency is higher than some chemical or physical transformation methods, and can stably inherit and express, with a genetic transformation efficiency of up to 30%-50%. The present invention can also transform a variety of quinoa cell types, including explant cells such as leaves, stem segments, and cotyledons, and can provide diverse transformation options for different genetic improvement targets, thereby expanding the scope of application of transformation. During Agrobacterium rhizogenes transformation, T-DNA is usually inserted into the quinoa genome in a relatively precise manner, rarely causing large-scale genome rearrangements or mutations, which is conducive to maintaining the stability of the quinoa genome and reducing the adverse effects on the normal growth and development of plants.

[0019] The RUBY gene appears red after expression. After the gene is successfully transferred from Agrobacterium to the quinoa genome, the quinoa roots exhibit a red phenotype. After amplification and identification of exogenous DNA fragments on the genome and RNA identification at the transcriptional level, and phenotypic identification of the offspring strains, it was found that the product finally prepared by the method of the present invention not only has a high transformation frequency, but can also introduce large fragments of DNA, and the fragments introduced into plant cells are accurate; at the same time, the number of imported gene copies is low, mostly only 1-3, the expression effect is good, and it can be stably inherited, and most of them conform to the Mendel's law of inheritance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a diagram showing the effect of quinoa rooting transformation in the present invention; Figure 2 This is a diagram showing the expression of the RUBY gene in quinoa hairy roots in the present invention; Figure 3 This is a diagram showing the results of semi-quantitative PCR detection of root transformation in the present invention; Figure 4 This is the result of the quantitative PCR detection of root transformation in the present invention; Figure 5 This is a diagram showing the expression of exogenous genes using the method of the present invention. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Agrobacterium is a Gram-negative bacterium that transforms host cells by transferring a specific DNA fragment from a tumor-inducing (Ti) plasmid into the host cell genome. In the natural environment, the transferred DNA (T-DNA) carries a suite of oncogenic and opine-metabolizing genes. Their expression in plants can induce tumor formation in infected tissues and synthesize opines, which serve as a nitrogen source for the bacteria. Molecular cloning techniques can replace T-DNA with the target gene, making Agrobacterium a natural tool for effectively introducing foreign genes into plants.

[0028] The Agrobacterium rhizogenes transformation method of quinoa in the present invention comprises the following steps: (1) Seed treatment: Sodium hypochlorite disinfection, germination on 1 / 2MS (3% sucrose) medium for 3-4 days; (2) Pre-transformation operation: Cut the hypocotyl of quinoa seedlings with a sterilized blade and culture the cut shoots on 1 / 2MS (3% sucrose) medium for 4-5 days; (3) Transformation: Immerse quinoa seedlings in LB liquid medium containing Agrobacterium rhizogenes and culture; (4) Dark protection treatment: Transfer the infected quinoa seedlings to sterilized filter paper to absorb the bacterial liquid on the surface, and then transfer them to 1 / 2MS (3% sucrose) medium covered with a layer of sterile filter paper and keep them away from light for two days; (5) Screening and cultivation of positive strains: After the light-proof treatment, the quinoa seedlings can continue to be cultured on the culture medium, or the infected quinoa seedlings can be transferred to the soil for culture. Positive roots will begin to grow in about two weeks.

[0029] The above-mentioned 1 / 2MS (3% sucrose) medium was purchased from Coolbo Company. Its specific components are shown in Table 1 below:

[0030] The present invention will be described in detail below by way of examples.

[0031] Example 1 The method for transforming quinoa with Agrobacterium rhizogenes in this embodiment comprises the following steps: (1) Seed treatment: Sodium hypochlorite disinfection, germination on 1 / 2MS (3% sucrose) medium for 3 days; (2) Pre-transformation operation: Cut the hypocotyl of quinoa seedlings with a sterilized blade, and culture the cut shoots on 1 / 2MS (3% sucrose) medium for 4 days; (3) Transformation: Immerse quinoa seedlings cultured for 4 days in a culture medium containing Agrobacterium rhizogenes and culture at 28°C and 200 rpm for 20 minutes; (4) Dark protection treatment: Transfer the infected quinoa seedlings to sterilized filter paper to absorb the bacterial liquid on the surface, and then transfer them to 1 / 2MS (3% sucrose) medium covered with a layer of sterile filter paper for 2 days and dark protection for 2 days; (5) Screening and cultivation of positive strains: After the light-proof treatment, the quinoa seedlings can be cultured on the culture medium until positive roots grow. Figure 1 As shown in FIG, the expression of the RUBY gene in the positive strain obtained is as follows Figure 2 shown.

[0032] Example 2 This example mainly verifies the genetic stability of the specific transformation method in Example 1. The transformed quinoa plants are propagated for multiple generations, and positive transgenic lines of the T1 generation are obtained by screening and identification. The seedlings of about one week old are transplanted to the soil and cultured for 3-4 months, and then the seeds are harvested to obtain the T2 generation. The T2 generation is planted and phenotypic identification is performed, such as Figure 5 As shown, it was finally observed that the exogenous gene could be stably inherited to offspring, proving that the transformation system of the present invention is reliable and practical.

[0033] The above experiments ultimately revealed that by introducing stress-resistance genes into quinoa, drought-tolerant genes were introduced. Comparative tests under drought conditions showed that the transgenic quinoa outperformed non-transgenic quinoa in terms of survival rate, biomass, and yield, confirming its enhanced drought tolerance. Similar results were also observed in tests under other stressful conditions, such as saline-alkali and low temperatures.

[0034] Genes rich in essential amino acids or other nutrients were transferred into quinoa. Testing showed that the content of corresponding amino acids or nutrients in genetically modified quinoa seeds increased significantly. For example, the lysine content could be increased by 20%-30%, proving its improved nutritional value.

[0035] By introducing genes related to growth and development, such as genes that promote root development, observation and comparison found that the root system of transgenic quinoa was more developed, and indicators such as root length, root volume and root dry weight were better than the control. The overall growth potential of the plant was better, and the plant height and stem thickness also increased, indicating that the growth characteristics were optimized.

[0036] Genes related to the synthesis of secondary metabolites were introduced, and tests found that the content of target secondary metabolites in transgenic quinoa was significantly increased. For example, the content of certain flavonoids could increase by more than 50%. These secondary metabolites have antioxidant and antibacterial activities, which can enhance quinoa's own defense capabilities and potential medicinal value.

[0037] Example 3 In this example, the obtained positive strains were identified at the DNA level. The root genomic DNA of the negative control Q75, the positive control K599 and the positive strains were extracted respectively to detect whether the rolB and rolD genes in the Agrobacterium Ri plasmid were successfully integrated into the quinoa chromosome. Figure 3 As shown, the gene expression of two successfully transformed positive strains at the RNA level was detected. Figure 4 As shown in the results, it was finally found that at the genomic level, the rolB and rolD genes in the Agrobacterium Ri plasmid were successfully integrated into the quinoa chromosome; at the transcriptome level, the target genes were expressed.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A method for transforming quinoa with Agrobacterium rhizogenes, characterized in that: The following steps are involved: (1) sterilizing quinoa seeds and then culturing and germinating them on a culture medium to obtain quinoa seedlings; (2) Cutting parts of quinoa seedlings and culturing them on a culture medium; (3) After culturing in step (2), immersing the culture medium containing Agrobacterium rhizogenes for culturing; (4) After culturing in step (3), the cells are transferred to sterilized filter paper to absorb the bacterial liquid on the surface, and then transferred to a culture medium covered with a layer of sterile filter paper for culturing; (5) The quinoa plants cultured in step (4) are screened to obtain the obtained product.

2. The method for transforming quinoa with Agrobacterium rhizogenes according to claim 1, wherein In step (1), the disinfection treatment is performed using sodium hypochlorite, the culture medium is 1 / 2MS culture medium containing 3% sucrose, and the culture time is 3-4 days.

3. The method for transforming quinoa with Agrobacterium rhizogenes according to claim 1, wherein In step (2), the cutting is performed by cutting the hypocotyl of the quinoa seedling with a sterilized blade, and the culturing is performed by culturing the cut shoot part on a 1 / 2 MS medium containing 3% sucrose for 4-5 days.

4. The method for transforming quinoa with Agrobacterium rhizogenes according to claim 1, wherein In step (3), the culture medium containing Agrobacterium rhizogenes is LB liquid culture medium, and the culture is carried out at 28° C. and 200 rpm for 20 minutes.

5. The method for transforming quinoa with Agrobacterium rhizogenes according to claim 1, wherein In step (4), the culture medium is 1 / 2MS culture medium containing 3% sucrose.

6. The method for transforming quinoa with Agrobacterium rhizogenes according to claim 1, wherein: In step (5), the screening is to detect whether the transformed specific gene is expressed.

7. A quinoa plant obtained by the quinoa transformation method using Agrobacterium rhizogenes according to any one of claims 1 to 6.

8. Use of the quinoa plant according to claim 7 in gene function research and molecular breeding of quinoa.

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